Steroid induced exocytosis: The human sperm acrosome reaction

Vol. 160, No. 2, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 828-833

April 28, 1989

STEROID

INDUCED

Richard A. Osmanl”,

EXOCYTOSIS:

THE HUMAN SPERM ACROSOME

Matthew L. Andria*#, A. Daniel Joness,

REACTION

and Stanley Meizells

‘Department of Human Anatomy *Department of Medical Microbiology and Immunology aFacility for Advanced Instrumentation University of California Davis, CA 95616 Received

March

21,

1989

By a combination of organic precipitation and high pressure liquid chromatography, human sperm acrosome reaction inducing activity has been purified from the fluid aspirated from preovulatory human ovarian follicles and identified as 4pregnen-3,20-dione (progesterone) and 4-pregnen-17ol:ol-3,20-dione (17ahydroxyprogesterone). It is argued that progesterone is present at the site of fertilization of placental mammals in concentrations sufficient for activity, and hence provides a mechanism of 0 1989 Academic Press, Inc. inducing the acrosome reaction, an exocytotic event, in vivo. In several biological systems steroids have been shown to elicit a rapid response mediated at the cell surface independent of macromolecular synthesis (l), the classically held mechanism of steroid action. A well studied example is the progesterone induced maturation of meiotically arrested -pus &t& eggs (2,3) Here we describe a rapid steroid induced exocytosis. The mammalian sperm acrosome is a secretory organelle that is formed during the late phase of spermatogenesis when golgi derived vesicles fuse and coalesce around the anterior portion of the spermatid nucleus. The acrosome reaction involves the fusion of the outer aspect of the acrosomal membrane with the overlying plasma membrane at multiple sites, releasing hybrid vesicles and exposing the acrosomal contents and the inner acrosomal membrane to the extracellular milieu (4,5). This modified exocytosis can be induced only after the sperm have undergone a post*Present address: Dept. Biology, College of Alameda, Alameda CA 94501. #Present address: Dept. Oncology, Stanford University, Stanford CA 94305. sTo whom reprint requests should be addressed. Abbreviations HFF, preovulatory human ovarian follicular fluid; HPLC, high pressure liquil chromatography; BSA, bovine serum albumin; HSA, human serum albumin; v/k volume/volume; S.D., standard deviation; DMSO. dimethyl-sulfoxide. 0006-291X/89 $1.50 Copyright 6 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ejaculatory maturation known as capacitation. Capacitation normally occurs within the female reproductive tract but can be mimicked by culture in vitro (4). The acrosome reaction seems required for both the sperm’s penetration of the zona pellucida, a glycoprotein coat surrounding mammalian oocytes, and fusion with the oocyte plasma membrane (4). The identification of the physiological site(s) and initiator(s) of the acrosome reaction has been a matter of contention (5). Human sperm acrosome reaction inducing activity, henceforth activity, has been found in the fluid aspirated from preovulatory human ovarian follicles (6,7). Two components of that activity have now been purified and identified as 4-pregnen-3,20-dione (progesterone) and 4pregnen-17cz-ol-3,20-dione (17a-hydroxyprogesterone). MATERIALS

AND METHODS

Sperm incubation medium was a bicarbonate buffered medium (7) with 26 mg/mI BSA (Sigma #A7030). The HSA medium used for steroid dilution was identical to sperm incubation medium except HSA (Sigma #A3782) - to mimic the presence of HSA in HFF - was substituted for BSA. HFF was obtained from women participating in the in vitro fertilization program of the Northern California Fertility Center (Sacramento, CA). To induce maturation of multiple follicles, women were treated with gonadotropins (7). Follicle maturation in such stimulated cycles appears normal in terms of morphology, cellular composition, and biochemistry (8,9). Batches were made by thawing and pooling samples from at least nine women. HFF was fractionated by stirring with 9 volumes of acetonitrile followed by centrifugation at 10,000 g for 10 min. The resulting supernatant fluid was dried by rotary evaporation and reconstituted with water to l/10 original HFF volume. For additions to cell cultures the 1 Ox extract was diluted back to lx with HSA medium (above). Aliquots (0.1 ml) of the 1Ox extract were further fractionated by reverse (see Fig. 1 legend) and normal (Vydac silica column #lOl HS54; 100% chloroform to 100% methanol linearly over 40 min) phase HPLC. Human sperm were obtained by masturbation from healthy donors selected by semen quality: sperm count, motility, and morphology. All results represent experiments with at least three different donors on different days. A >95% motile morphologically homogeneous population of sperm was obtained by centrifuging semen through a discontinuous Percoll gradient; the resulting sperm pellet washed by centrifugation, resuspended to 6 x 10s cells/ml with incubation medium, and capacitated by incubation in 200 pl cultures for 26-30 hrs at 37 “C (7). Following treatments with HFF, fractions thereof, or commercial steroids, sperm cell cultures were incubated an additional 10 min during which motility assessments were made. Cultures were then immediately fixed and immuno-stained with an antibody to the acrosomal matrix (8) to visualize and count the percentage acrosome reacted cells. All counting was done experimentally blind. It should be noted that the acrosome reaction assay saturates at about 40-50%; i.e. just under half of the sperm population is detected as acrosome reacted, or by inference, capacitated. Ten percent HFF is saturating as increasing the HFF concentration to 30% does not increase the size of the responsive population. From 10 to 20% of the cells of untreated or control cultures are identified as acrosome reacted. Results herein are of course limited by the range of this assay. RESULTS HFF was fractionated assays with the reconstituted

by precipitation with acetonitrile. Acrosome reaction supernatant fluid showed activity levels comparable to 829

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Vol. 160, No. 2. 1989

that of the native fluid (e.g. n = 3, mean % acrosome reactions St SD: 10% v/v HFF, 48 + 10; 10% v/v extract, 52 f 6; negative control, 13 f 3; with no qualitative nor quantitative change in motility - invariably exceeding 90% - after additions). Neither resuspensions nor reextractions of the precipitate yielded activity. Aliquots of this extract were further fractionated by reverse phase HPLC (Fig l), and activity was consistently found in only two fractions with retention times of 24 min (RT24) and 30 min (RT30). Ultraviolet absorbance scans revealed absorbance maxima at 243 nm for the baseline separated peak corresponding to each fraction. Repeated ‘HPLC runs generated a large pool of RT30 for further analysis. Gas chromatography-electron ionization mass spectrometry (VG Analytical ZAB-HSPF mass spectrometer) detected a dominant compound of molecular weight of 314 with a fragmentation pattern matching progesterone (Fig 2). Identification of this compound was further confirmed by proton nmr spectroscopy (Varian Instrument NM 500 Spectrometer) and HPLC coelution of rechromatographed RT30 with progesterone (Sigma #PO1 30) under both reverse and normal phase (10 min. retention time) conditions. Several progesterone derivatives were chromatographed by HPLC, and 17a-hydroxyprogesterone (Sigma #H5752) was found to coelute with twice-chromatographed RT24 under both reverse phase and normal phase (14 min. retention time) conditions. Resuspending all HPLC fractions in water provided convenient additions to cell cultures but quantitatively erratic suspensions of less polar fractions. Hence, the activities of commercial progesterone and 17a-hydroxyprogesterone were compared under more quantitatively controlled conditions: 1 ug/ml of either steroid was found to be saturating, eliciting acrosome reactions in 30% of the sperm (Table 1). Neither

0 4x

__.-

Omin.

10

20

__.-

.:._--_.-__.__-. ._-. _..__-__-__.-. RT30 _.-A

30

40

Figure 1 Acetonitrile extract of HFF was fractionated on a C-18 column (Vydac 201TP54, 250 x 4.5 mm) by reverse phase HPLC. A continuous gradient was used: 100% water to 100% acetonitrile over 40 min at a constant 1 ml/min flow rate. Fractions were dried in an evacuated centrifuge, resuspended in water to several concentrations, and assayed for activity by adding to capacitated sperm cultures such that aqueous additions did not exceed 20% final volume. 20% aqueous additions had no effect on the positive (cultures treated with follicular fluid) and negative controls which were assayed in parallel. 830

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Text Peak1

30. 25. 20_ IS. 4;

, 800 13-24

c 200 3.21

400 6:42

SCAN TIllE

600 lo:03

, , 1000 16:45

Peak1 ‘Xl ‘0

LIEfITSl113~ PREGN-4-ENE-3,20-OIONE, ~9.BETR.,IO.RLPHR.lII3272755-10-4 C21.H30.02. Bpk: 124ilut: 314 +x1‘0

Figure 2 Gas chromatograph and electron matched library mass spectrum.

ionization

mass spectrum

of RT30

and

increased concentrations (up to 20 pglml), combinations of the two progesterones (again up to 20 pg/ml each), nor supplementing the progesterones with other HPLC fractions provided significantly enhanced responses. 831

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table 1

Treatment

Final Concentration

% Acrosome Reactions

progesterone

1 pg/ml

30 f 6b

17a-hydroxyprogesterone

1 pg/ml

3oflb

HFF

IO % v/v

41*7o

negative control

14f5a

Steroids were disolved in DMSO then diluted with the bicarbonate buffered medium supplementedwith 26 mg/ml human serum albumin (HSA) to a 5% (v/v) final DMSO concentration. Aliquots of these solutions were added to capacitated sperm cultures to desired final steroid concentration with a final DMSO concentration of 0.5%. The negative control received a comparable DMSO-HSA solution and the HFF treated cultures were similiarly supplemented to 0.5% DMSO. Under these conditions, no quantitative nor qualitative change in motility was detected after any of the reported additions ( >90% for experiments reported here). Progesterone induced acrosome reactions were found to be morphologically normal as judged by electron microscopy (7). Five replicates were carried out for each treatment. Means with same superscript were not significantly different (w.05) by ANOVA.

DISCUSSION Cumulus cells surround the human egg at the time of fertilization

(12) Since

progesterone secretion by single human egg-cumulus complexes has been measured at roughly 5 to 15 ng/hr (13,14), the concentration of progesterone within the cumulus matrix should easily exceed the 1pg/ml (1 ngipl) concentration shown to be effective in these in vitro studies, more so if the cumulus matrix provides a significant barrier to diffusion of progesterone-protein complexes. Indeed, single human cumulus cell matrices (6) and fragments thereof (15) have been shown to produce acrosome reaction inducing activity in vitro. The present report has uncovered a plausible mechanism for both the HFF and human cumulus induced acrosome reaction. The zona pellucida of a number of mammals can induce acrosome reactions (4). It should be clear however, that in order for the zona pellucida to have a role in inducing the acrosome reaction in vivo, the fertilizing sperm must maintain an intact acrosome as it passes through the cumulus cell matrix which surrounds the eggs of most mammals at the time of fertilization (4). These cells have already been shown to secrete progesterone in many species (16) and independently, to produce acrosome reaction inducing activity in at least two species (6,15,17). Further, acrosome reacted sperm have been shown to be capable of binding to the zona pellucida in at least three mammalian species in vitro (18,19,20). Progesterone may provide a general mechanism of inducing the sperm acrosome reaction in placental mammals. The HFF induced human sperm acrosome reaction is a rapid event (11) initiated within ten seconds by an influx of extracellular Ca*+ (21). Since a Ca*+ influx appears to be a requirement for acrosome reactions in ail species studied no matter 832

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what stimulus is used (4, 20, 21, 22), the present findings suggest that progesterone is a/the causative agent mediating the rapid HFF induced Can+ influx. The rapidity of this exocytotic event excludes a mechanism based on alterations in macromolecular synthesis and hence provides a potentially novel mechanism of steroid action. ACKNOWLEDGMENTS Supported by NIH grant HD-06698 (to SM) Foundation grant GA-PS-8529 (to SM). We thank J. spectra, B. King and G. Fry for electron microscopy, K. sperm, HS21, and P. Thomas, A. Bottini, S. Chaykin, E. technical advice.

and in part by Rockefeller deRopp and S. Toto for nmr Bechtol for antibody to human Friedrich, and RScibienski for

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